The last major advance in the treatment of metastatic bladder cancer (BC) took place in 1997 with the advent of gemcitabine. Despite this advance, visceral metastases are usually fatal. The overall goal of the proposed studies is to develop small molecule inhibitors that block a critical node in the metastatic process. We found that Rai GTPases serve as the molecular switches of a therapeutically tractable signaling pathway that allows UC cells to grow in the lung, the most common visceral metastatic site. The clinical significance of this pathway and validity of Rai as a therapeutic target is supported by finding that high Rai expression in tumors places patients at higher risk for metastasis and the requirement of Rai expression for lung metastasis to occur in animal models of UC. Our Guiding Hypothesis for this application is that small molecules targeting Rai provide effective therapy for metastatic UC. With support from the MD Anderson Bladder SPORE Developmental Research Program (DRP), we evaluated >500K compounds for their ability to bind RalA or RalB in computational and combinatorial screens and selected 99 hits. These were evaluated in a series of secondary assays allowing us to select Rai Binding Compound (RUC)8 and 10 to be pursued in this application. RUC8 and 10 were selected because they: 1) inhibit RalA to RalBPI binding in human UC cells and RalA induced spreading in murine embryo fibroblasts; 2) inhibit in vitro monolayer growth (IC50 0.5-1.9 pM) of human UC cells; 3) bind RalB directly by nuclear magnetic resonance (NMR) spectroscopy; and 4) have good pharmacokinetic (PK) properties in mice (Cmax 1.3-23 pM, T1/2 3.7-4.6 hrs). To develop this novel class of agents we propose the following Specific Aims: Aim 1: Characterize higher potency 2^* generation compounds based on RUC8 and 10 using medicinal chemistry, computational fragment-based design, and similarity search of chemical databases. In the unlikely situation that higher potency compounds are not found in Aim 1, we will pursue Aim 2 and 3 using RUC8 and 10, given their adequate IC50 and in vivo PK. Aim 2: Evaluate 2 generation compounds for their in vivo therapeutic efficacy in novel human UC models of visceral metastasis. Aim 3: Develop predictive biomarkers of response to antlRal therapeutics in human tissues that will position us for Phase 1 trials by end of this project. Documented interest by Astra Zeneca in our work improves overall chances for success in translating our novel Rai inhibitors into the clinical setting as anticancer therapeutics.